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- EMDB-6335: Three-Dimensional Reconstruction of Lipid Nanodisc Reconstituted ... -

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Basic information

Entry
Database: EMDB / ID: EMD-6335
TitleThree-Dimensional Reconstruction of Lipid Nanodisc Reconstituted Yeast V-ATPase Membrane Sector
Map dataReconstruction of Lipid Nanodisc Reconstituted Yeast V-ATPase Membrane Sector
Sample
  • Sample: V-ATPase membrane sector (Vo) isolated from yeast membranes and reconstituted with lipid nanodisc
  • Protein or peptide: Vacuolar-type (V-) ATPase membrane sector (Vo)
  • Protein or peptide: MSP1E3D1
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / synthetic construct (others)
Methodsingle particle reconstruction / negative staining / Resolution: 16.3 Å
AuthorsStam NJ / Wilkens S
CitationJournal: J Biol Chem / Year: 2017
Title: Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel: DEFINITION OF THE INTERACTION OF ROTOR AND STATOR AND IMPLICATIONS FOR ENZYME REGULATION BY REVERSIBLE DISSOCIATION.
Authors: Nicholas J Stam / Stephan Wilkens /
Abstract: Eukaryotic vacuolar H-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V-ATPase and membrane-integral ...Eukaryotic vacuolar H-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V-ATPase and membrane-integral V proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast V The structure indicated that, when V is released from V, the N-terminal cytoplasmic domain of subunit a (a) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the a-d interface. Here we reconstituted V into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between a and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the a-d interaction, as shown by isothermal titration calorimetry and gel filtration of recombinant subunits. A recent cryo-EM study of holo V-ATPase revealed three major conformations corresponding to three rotational states of the central rotor of the enzyme. Comparison of the three V-ATPase conformations with the structure of nanodisc-bound V revealed that V is halted in rotational state 3. Combined with our prior work that showed autoinhibited V-ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V and V functions to prevent unintended reassembly of holo V-ATPase when activity is not needed.
History
DepositionMay 6, 2015-
Header (metadata) releaseMay 27, 2015-
Map releaseMay 11, 2016-
UpdateMay 25, 2016-
Current statusMay 25, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6335.gif

Downloads & links

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Map

FileDownload / File: emd_6335.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Lipid Nanodisc Reconstituted Yeast V-ATPase Membrane Sector
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.5 Å/pix.
x 96 pix.
= 336. Å		3.5 Å/pix.
x 96 pix.
= 336. Å		3.5 Å/pix.
x 96 pix.
= 336. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.18370923 - 0.34209979
Average (Standard dev.)0.00211898 (±0.02535874)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z336.000336.000336.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.1840.3420.002

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Supplemental data

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Sample components

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Entire : V-ATPase membrane sector (Vo) isolated from yeast membranes and r...

EntireName: V-ATPase membrane sector (Vo) isolated from yeast membranes and reconstituted with lipid nanodisc
Components
  • Sample: V-ATPase membrane sector (Vo) isolated from yeast membranes and reconstituted with lipid nanodisc
  • Protein or peptide: Vacuolar-type (V-) ATPase membrane sector (Vo)
  • Protein or peptide: MSP1E3D1

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Supramolecule #1000: V-ATPase membrane sector (Vo) isolated from yeast membranes and r...

SupramoleculeName: V-ATPase membrane sector (Vo) isolated from yeast membranes and reconstituted with lipid nanodisc
type: sample / ID: 1000 / Number unique components: 2
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: Vacuolar-type (V-) ATPase membrane sector (Vo)

MacromoleculeName: Vacuolar-type (V-) ATPase membrane sector (Vo) / type: protein_or_peptide / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4743 / synonym: Yeast
Molecular weightTheoretical: 300 KDa

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Macromolecule #2: MSP1E3D1

MacromoleculeName: MSP1E3D1 / type: protein_or_peptide / ID: 2 / Name.synonym: membrane scaffold protein, nanodisc / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 30 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET28a

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.04 mg/mL
BufferpH: 7.4 / Details: 20 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA
StainingType: NEGATIVE
Details: 2% w/v uranyl formate applied to grids with adsorbed protein
GridDetails: 200 mesh copper grid with thin carbon support, glow-discharged in air
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
DateNov 13, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 390 / Average electron dose: 17 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 85800 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.08 µm / Nominal defocus min: 1.125 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL

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Image processing

DetailsParticles were selected using a monitored semi-automated selection program.
CTF correctionDetails: By micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.3 Å / Resolution method: OTHER / Software - Name: EMAN2, Relion / Number images used: 47422
FSC plot (resolution estimation)

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